Activated enzyme compositions



added to them. tivity in the presence of chemicals.

cal additives.

United States Patent tion of New Jersey No Drawing. Filed Nov. 25, 1957, Ser. No. 698,366

16 Claims. (Cl. 195-68) This invention relates to the activation of proteolytic enzymes.

In recent years a considerable amount of research effort has been devoted to the study of a class of biological catalysts commonly known as enzymes. Notable advances have been made in this field and vast quantities of scientific data have been accumulated regarding physiological and chemical properties of enzymes. A number of them have been prepared in highly purified form and their chemical constitution has been studied in detail. It has been established, for example, that enzymes are largely protein in nature; that their essential structural unit is a long chain arising from the condensation of a number of amino acid residues joined through peptide linkage; that they are large size molecules of high molecular weight; and that, in solution, they migrate characteristically in an electrical field.

Because enzymes are not as ble, their characteristics are usually established along functional lines. Thus, it is typical of some enzymes that they are, under optimal conditions of temperature, either inhibited or not aifected at all when chemical agents are Others demonstrate an accelerated acyet structurally identifiazymes are completely inactive in the absence of chemi- Agents which are capable of increasing the activity of such weakly active or inactive enzymes are called activators.

The search for suitable activators has been considerj ably intensified'in recent years due to the discovery that f proteolytic enzymes are useful in the medical field in the physiological debridement of eschar, blood and pus. Al-

though a large number of proteolytic enzymes are available, laboratory tests show that their proteolytic activity is weak and slow in onset, thus reducing their applicability from an industrial point of view.

It has now been found that certain proteolytic enzymes are activated inthe presence of ions of calcium, magnesium, manganese, cobalt or combinations thereof.

Preferred for use as single activators are calcium and magnesium. Where a combination of two metals is employed to activate the enzyme, calcium and magnesium I are preferred. The proportions of the metal ions to be used in activating proteolytic enzymes according to this invention may be varied considerably, depending upon the number of different activating ions employed, the pH of the final product desired and other factors. Satisfactory results are obtained when the ions are added in a non-toxic concentration from about 0.01 molar to about 1.0 molar. For optimal activation, it is preferred to use a concentration from about 0.05 molar to about 0.1 molar.

Useful non-toxic salts of the metal ion activators include chlorides, acetates, lactates, propionates, gluconates and, preferably, phosphates. The addition of alkali In fact, some enmetal ions, i.e. potassium and sodium, preferably in the form of phosphates or acetates, is advantageous primarily for the purpose of buffering the enzyme compositions. For topical use on the animal organism it has been found that adjustment of pH from about 3.0 to about 5.0, optimally from about 3.0 to about 4.0 is satisfactory. Further, it is also desirable to include a reducing agent such as a thiosulfate, i.e. sodium or potassium or, preferably, cysteine in order to obtain maximum results. The inclusion of cysteine in a concentration from about 0.05 molar to about 0.1 molar is satisfactory.

The term proteolytic enzyme as used herein is meant to include pinguinain, papain, ficin, bromelin, bromelain and asclepain. Preferred for use as the proteolytic enzyme is pinguinain or ficin; especially preferred is pinguinain.

Pinguinain as used in this invention is meant to include the proteolytic enzyme described by Asenjo (Science, v. 95, No. 2454, pp. 48-49) as well as the improved form of pinguinain obtained by macerating the fruit of Bromelia pinguin, separating the juice from the fibrous debris, spray-drying and, if desired, further purifying by precipitation with ammonium sulfate, aqueous methanol, aqueous ethanol or aqueous isopropanol. Pinguinain is a high molecular weight enzyme system comprised essentially of three components, two of which (A and B) are proteinaceous in character and readily characterizable by their mobilities in an electrophoretic field, the third (C) being non-protein and lacking in electrophoretic mobility between pH 3.5 and pH 6.0. The average electrophoretic mobility in 0.1 ionic glycine buffer at pH 3.5 for A component is 0.377, for B componeut it is 0.234. The average mobility in acetate buffer at pH 4.6 for A component is 0.175, for B component it is 0.064. The isoelectric point for A component is above pH 6.0, for B component pH 5.25. .The enzyme demonstrates optimal proteolytic activity (in an Azocoll substrate) at a pH from about 5.2 to about 5.5 and at pH 7.3, and is inactivated at temperatures in excess of C. It is not activated by ascorbic acid, but is activated by sodium thiosulfate and cysteine. It is soluble in water, normal saline and aqueous salt solutions of low ionic strength.

In actual practice, the activated enzymes of this invention may be used in the form of aqueous solutions either for topical administration or for intramuscular injection. They may also be used in the form of gels suitable for application on burned or ulcerated areas. The quantity about 0.01% to about 1%, preferably about 0.5% en zyme on a weight basis, or from about 1,000 to about 20,000 Azocoll units per milliliter. Similar proportions may be employed when any one of the other enzymes specified hereinabove is formulated. To ensure maximum proteolytic activity, activated enzyme solutions and gels for topical use should be adjusted to a pH from about 3.0 to about 5.0 with an appropriate buffer, preferably acetate or phosphate. Solutions intended for intramuscular injection should be adjusted to near neutrality. The utility of this material for use in human therapy has not as yet been clinically established.

Among the substances suitable for formulation of activated enzyme gels there may be mentioned vegetable gums such as acacia, tragacanth, ghatti or karaya; mucilages such as those obtained from guar, bean, linseed, the locust bean or other related leguminous plant seeds; or cellulose derivatives such as carboxymethylcellulose, methylcellulose, starch or starch derivatives. It is to be understood that altliough'the activated enzyme compositions of this invention may find primary application as debriding and anti-inflammatory agents, they are also useful in a wide variety of industrial processes where proteolytic enzymes are conventionally employed, as for example in chill-proofing of beer, cheese-making, leather manufacture, as meat .tcnderizers, etc., and it is intended that these applications be included within the scope of this invention.

The Azocoll unit as used herein is an index of proteolytic activity as determined by the method of Bidwell (Biochemical Journal, 46, pp. 589-598, 1950) and Oakley et al. (Journal of Pathology and Bacteriology, 58, pp. 229235, 1946).

Enhancement of proteolytic activity in the presence of metal ions is demonstrable by means of tests on guinea pig eschar. The procedure is as follows: The hair is removed from a guinea pig back with an animal hair clip- *vati'ons of the percentageof eschar Value Description No activity.

Separation of layers with little or no digestion.

. Separation of layers and some digestion.

Fair diglcstion with few small pieces broken ofi the main body of esc ar.

Good digestion with the eschar partially disintegrated.

Escher completely disintegrated with a few small particles remaining.

Eschar completely disintegrated.

The activating eifect of metal ions on proteolytic enzymes is also evidenced by in vivo tests on guinea pig eschar. The procedure is as follows: Hair is removed from a guinea pig back with an animal hair clipper. The animal is then injected with sodium pentobarbital. The animals back is then immersed in a water bath-'at'70" C. for 45 seconds. A burned areaof about two to three inches in diameter-is formed as a result of this immersion. Seven days after the burn, the animal is treated with the enzyme preparation, applied 'in ointinent form to the burned area on three successive days. The ointment is covered with a film'which is kept-inplace-b'y tape. Obserwhich was debrided on each animal tested are recorded each day "that'the bandages are removed. The bandage is'r'emoved an'd'the eschar is observed on thefirstfsecond and third days "after the initial application of enzyme. The ointment employe'din these testseonsiste'rl of 3 gfiof guarguin, 1.8 g. of pinguinain and 30ml. of'water. The enzyme 'andgum were mixed in a'drystate and'added to'the water immediately before use.

Results of in vivo tests onguinea pig'escharemploying pinguinain are given below. The fi'gure"percent debridement represents the percentage of the'eschar which was debrided on the'animal tested. Complete cleaning of'the per. The animal is then injected with sodium pentodt h b1 f d 1007 barbital. The animals back is then immersed in a water Woun 0 e vla e gra t tlssue 1S cons ere .3.

O bath at 70 -C. f0r 45 seconds. A burned area of about Amount. Percent debridement 2 to 3 inches in diameter is formed as a result of this ofenz yi ie immersion. Seven days after the animal has been g i 1 3 n h animal Dilucnt I blll'l'lci'lt is sacrificed and th eschar is hari ested. T e in tom 7 24 hrs 48 hrs. 72 hrs eschar is removed from the animal with a scalpel and treatment stored in the frozen state until needed for use. Immedi- $322 ateiy before use, while it is still in the frozen state, the I escha-r is cut into pieces 1 X 1 cm. for a 5 ml. volumc'of 220x10 Acetate 50 reaction mixture; 1 x 2 cm. for a 10 ml. volume of re- 3% action mixture, and l x 3 cm. for a 15 ml. volume of ,3 reaction mixture. The eschar pieces are permitted to 420x10 Watei' 5 5 e5 thaw about vl0 minutes before they are placedun the 381x103; MM calcium cmmdemetate ,5 ,90 100 reaction mixtures. The reaction mixture containing a butter. g: 100 piece of eschar is incubated in an oven at 37 C. for be- 381mm calcium chloride 32 tween 16 and 18 hours. The eschar is then inspected Phata bllfier' l 3g 3g visually and the activity of the preparation is evaluated. 55 381x10 oli a magn sium chloride lies '5 97 100 'lgte term reaction mixture asl used in these 'gests means 381x10, Z E L' cboridley 5 g3 32 533 t mixtur o. "si 0 sciar u st ate t enz e magnesium w P 9 100 the e S b r e ym 1 phosphate butler. i 25 99 100 ant. the particular carrier employed. 38l l0 0.05M calcium eligiridg, 0.95M 85 98 100 1 magnesium act p osp iate 75 '95 '100 Follow ng are the results of in vitro tests on guinea pig 60 phosphate bufien 50 95 l eschar using pinguinain:

Enzyme Azocoll Cone. of

c0110., value Buffer pH cysteine Metaliou Result: percent units/ml. (M)

6 285x10 3.4 0.008111 No 2 e 285x10 3.4 0. 008m 0.2M eobaiteliiondaunn 4 6 285 10 3.4 0. 008m 02M manganese ChlOIldtL- 3 0 285x10 2 3. 4 0. 008m 0.02M magnesium chloilde 5 6 285x10 3.4 0. 008m 0.2M cobalt'chlor'ide e 6 285 10 3.4 0.008m 0.2M manganese chloride 7 6 285x10 3.4 0. 008m 0.2M magnesium chloride '6 4 182 10 4.3 0.05 m 0.2M calcium chloride 7 10 27OX10 2 3.0 0. 008m 0.02M calcium chloride, 0.04M m. 6 10 270x10 Water 4.0 0.008m .do- 7 1o 270x10 Water-NaOH... 5.2 0. 008m do 5 10 270x10 o as 0. 008m 0.--- 2 The following tests illustrate the activating ability of metal ions on other proteolytic enzymes. The tests were conducted as follows: A 1 x 1 cm. piece of eschar was placed in each enzyme solution ml). The enzyme solution was prepared by weighing enzyme quantities and cysteine and adding 20 ml. of diluent activator, or buffer without activator as the case may be, to the weighed quantities of enzyme. One piece of eschar was added to 5 ml. of enzyme solution in a test tube. The reaction mixture was incubated in an oven at 37 C. for between 16 and 18 hours and the results of the test evaluated. The diluent activator used in these tests (and which may be employed in actual practice for the purposes specified hereinabove) had the following composition:

G. Calcium chloride 0.55 Magnesium acid phosphate 1.09 Disodium phosphate 0.057 Distilled water, q.s. 100.00 ml.

The results are as follows:

Enzyme Diluent Cysteine, pH Results 2g. papain- 0.1M sodium ace- 120 4. 3 Esehar partially tate pH 3.75. disintegrated. Do Dlluent activator- 120 4. 3 Eschar completely disintegrated. 2g. flcin-- 0.1M sodium ace- 120 4. 4 Escher partially tate pH 3.75. disintegrated. Do Diluent activator- 120 4. 4 Escher completely disintegrated.

What is claimed is:

1. An activated enzyme composition adjusted to a pH of from about 3.0 to about 5.0 comprising a plant proteolytic enzyme selected from the group consisting of pinguinain, papain, ficin, bromelin, bromelain and asclepain and, in non-toxic concentration, at least one metal ion selected from the group consisting of calcium, magnesium, manganese and cobalt in the form of their non-toxic salts.

2. A composition as set forth in claim 1 wherein the metal ions are contained in a concentration from about 0.01 molar to about 1.0 molar.

3. A composition as set forth in claim 1 wherein the metal ions are contained in a concentration from about 0.05 molar to about 0.1 molar.

4. An activated enzyme composition adjusted to a pH of from about 3.0 to about 5.0 comprising a plant proteolytic enzyme selected from the group consisting of pinguinain, papain, ficin, bromelin, bromelain and asclepain, at least one metal ion selected from the group consisting of calcium, magnesium, manganese and cobalt in the form of their non-toxic salts and at nontoxic concentrations, and a buffer.

5. A composition as set forth in claim 4 wherein the buffer is an acetate.

6. A composition as set forth in claim 4 wherein the buifer is a phosphate.

7. An activated enzyme composition adjusted to a pH of from about 3.0 to about 5.0 comprising a plant proteolytic enzyme selected from the group consisting of pinguinain, papain, ficin, bromelin, bromelain and as clepain, at least one metal ion selected from the group consisting of calcium, magnesium, manganese and cobalt in the form of their non-toxic salts and at non-toxic concentrations, a butter and cysteine.

8. An activated enzyme composition adjusted to a pH of from about 3.0 to about 5.0 comprising a plant proteolytic enzyme selected from the group consisting of pinguinain, papain, ficin, bromelin, bromelain and asclepain, at least one metal ion selected from the group consisting of calcium, magnesium, manganese and cobalt in the form of their non-toxic salts and at non-toxic concentrations, a butter and an alkali metal thiosulfate.

9. An activated enzyme composition adjusted to a pH of from about 3.0 to about 5.0 comprising a plant proteolytic enzyme selected from the group consisting of pinguinain, papain, ficin, bromelin, bromelain and asclepain, at least one metal ion selected from the group consisting of calcium, magnesium, manganese and cobalt in the form of their non-toxic salts and at nontoxic concentrations, a butler, cysteine and a pharmaceutical carrier.

10. A composition as set forth in claim 9 wherein the pharmaceutical carrier is an aqueous carrier.

11. A composition as set forth in claim 9 wherein the pharmaceutical carrier is a gel.

12. An activated enzyme composition adjusted to a pH of from about 3.0 to about 5.0 comprising pinguinain and, in non-toxic concentration at least one metal ion selected from the group consisting of calcium, magnesium, manganese and cobalt in the form of their nontoxic salts.

13. An activated enzyme composition adjusted to a pH of from about 3.0 to about 5.0 comprising pinuinain, at least one metal ion selected from the group consisting of calcium, magnesium, manganese and cobalt in the form of their non-toxic salts and at non-toxic concentrations and a buffer.

14. An activated enzyme composition adjusted to a pH of from about 3.0 to about 5.0 comprising pinguinain, at least one metal ion selected from the group consisting of calcium, magnesium, manganese and cobalt in the form of their non-toxic salts and at non-toxic concen trations, a buffer and cysteine.

15. An activated enzyme composition adjusted to a pH of from about 3.0 to about 5.0 comprising pinguinain, at least one metal ion selected from the group consisting of calcium, magnesium, manganese and cobalt in the :form of their non-toxic salts, a buffer and an alkaline metal thiosulfate.

16. An activated enzyme composition adjusted to a pH of from about 3.0 to about 5.0 comprising pinguinain, at least one metal ion selected from the group consisting of calcium, magnesium, manganese and cobalt in the form of their non-toxic salts and at non-toxic concentrations, a bufier, cysteine and a pharmaceutical carrier.

References Cited in the file of this patent UNITED STATES PATENTS Holmbacker Oct. 24, 1950 Damaskus Apr. 4, 1961 OTHER REFERENCES Sumner et al.: Chemistry and Methods of Enzymes, 3rd Ed., 1953, Academic Press, N.Y., pp. 166-198, pp. 187 and 189-191 esp. pertinent.

Grob: J. Gen. Physiology, 29:4, March 20, 1946, pp. 223, 227, 233 and 235-237.

Kimmel et al.: J. Biol. Chem., Vol. 207, pp. 515-531, 1954.

Hwang et al.: Ann. N.Y. Acad. 191-196, 204-207, May 16, 1951.

Gold et al.: Amer. J. of the Med. Sciences, Vol. 228, pp. 568-585, 1954.

Science News Letter, 70:20, Nov. 17, 1956, p. 312.

Heilesen: J. Inv. Dermatology, 23:1, pp. 7-15, July 1954.

Altemeier et al.: Annals October 1951.

Sherry et al.: Procs. of the Sec. for Exptl. Biol. and Med, 87:1, pp. -128, October 1954.

Chemical Abstracts (1), vol. 48, p. 5243g, 1954.

Chemical Abstracts (2), vol. 45, p. 7165i, 1951.

Sciences, 54:2, pp.

Surg. 134:4, pp. 581-583, 

1. AN ACTIVATED ENZYME COMPOSITION ADJUSTED TO A PH OF FROM ABOUT 3.0 TO ABOUT 5.0 COMPRISING A PLANT PROTEOLYTIC ENZYME SELECTED FROM THE GROUP CONSISTING OF PINGUINAIN, PAPAIN, FICIN, BROMELIN, BROMELAIN AND ASCLEPAIN AND, IN NON-TOXIC CONCENTRATION, AT LEAST ONE METAL ION SELECTED FROM THE GROUP CONSISTING OF CALCIUM, MAGNESIUM, MANGANESE AND COBALT IN THE FORM OF THEIR NON-TOXIC SALTS. 